Berhane Ghebrehiwet

Introduction: Heightened inflammatory and thrombotic processes are common hallmarks of vascular diseases. The interaction between these two processes remains unclear and a better understanding of these links can allow for the design of more effective treatment options. Activation of complement component 1 (C1) leads to the initiation of the classical arm of the complement cascade, availability of plasma C1q, and the potential association of C1q and receptors for C1q. The association of C1q and gC1qR, the receptor for the globular head of C1q, is notable and has been associated with a wide range of disturbed physiological processes. We have recently shown that when this interaction occurs on vascular wall cells, including adventitial fibroblasts and vascular smooth muscle cells, there is a significant up‐regulation of tissue factor (TF) expression. However, whether or not this TF is biologically active and can facilitate extrinsic coagulation activation remains unknown. We hypothesized that TF expressed via gC1qR‐C1q association would support the progression of extrinsic coagulation. Methods: We quantified the association of Factor VII/VIIa (FVII/FVIIa) with adventitial fibroblast and vascular smooth muscle cell TF, using colorimetric assays. Further, we observed the formation of Factor Xa and Factor IIa (thrombin), as well as the concentration of intracellular Akt (protein kinase B) and phosphorylated Akt. Results/Conclusions: Our results indicate that TF expression in response to C1q exposure accelerates zymogen formation within the extrinsic coagulation cascade and alters Akt/p‐Akt expression. Overall, these findings highlight a significant connection between altered innate inflammation and heightened thrombin generation.

The association between C1q deficiency and the development of Systemic Lupus Erythematosus (SLE) is well established. Several studies have shown that deficiency in C1q is associated with failed apoptotic cleanup, leading to SLE progression. However, the magnitude of this correlation indicates that C1q may play a much more complex role in the development of lupus. This study provides further insight into the pathogenesis of SLE by investigating the consequences of the interaction between C1q and CD4+ T-cells in the breakdown of self-tolerance. Since the C1q/C1q receptor interaction is postulated to play a role, we first confirmed the presence of surface-expressed C1q and C1q receptors on CD4+ T-cells. Then, cell proliferation assays were performed in the presence and absence of purified C1q, gC1qR, and cC1qR. The supernatants of these cultures were used to determine the levels of immunoregulatory cytokines released. Our data confirm that increasing concentrations of C1q and gC1qR significantly inhibited cell proliferation. Furthermore, the CD4+ cells treated with either C1q or gC1qR secreted reduced inflammatory cytokines, such as IL-6 and TNF-alpha, compared to the untreated controls, suggesting that C1q deficiency facilitates the uncontrolled secretion of these critical cytokines, thus contributing to SLE. Although the role of pro-inflammatory cytokines in the induction of SLE is well documented, the mechanism by which C1q contributes to the disease is still a study in progress. Our data demonstrate that the interaction between C1q and its receptors on CD4+ T cells plays a critical role in the suppression of pro-inflammatory cytokines that cause tissue injury in SLE. Therefore, the C1q-C1qR axis may provide a rationally sound target for the design of novel therapeutic approaches for SLE treatment.

The plaques associated with Alzheimer’s disease are formed as a result of the aggregation of Aβ peptides, which vary in length from 38 to 43 amino acids. The 1-40 peptide is the most abundant, while the 1-42 peptide appears to be the most destructive to neurons and/or glial cells in a variety of assays. We have demonstrated that aggregated Aβ, a state prior to plaque formation, will activate the plasma bradykinin-forming pathway when tested in vitro. Aggregation is zinc-dependent, optimal at 25–50 µM, and the rate of aggregation is paralleled by the rate of activation of the bradykinin-forming pathway as assessed by plasma kallikrein formation. The aggregation of Aβ 1-38, 1-40, and 1-42 is optimal after incubation for 3 days, 3 h, and under 1 min, respectively. The cascade is initiated by the autoactivation of factor XII upon binding to aggregated Aβ; then, prekallikrein is converted to kallikrein, which cleaves high-molecular-weight kininogen (HK) to release bradykinin. Studies by a variety of other researchers have demonstrated the presence of each “activation-step” in either the plasma or spinal fluid of patients with Alzheimer’s disease, including activated factor XII, kallikrein, and bradykinin itself. There is also evidence that activation is more prominent as dementia worsens. We now have medications that can block each step of the bradykinin-forming pathway as currently employed for the therapy of hereditary angioedema. Given the current state of therapy for Alzheimer’s disease, which includes monoclonal antibodies that retard the rate of progression by 30% at most and have significant side effects, it seems imperative to explore prophylaxis using one of the long-acting agents that target plasma kallikrein or factor XIIa. There is a long-acting bradykinin antagonist in development, and techniques to target kallikrein mRNA to lower levels or knock out the prekallikrein gene are being developed.

Understanding at the molecular level of the cell biology of tumors has led to significant treatment advances in the past. Despite such advances however, development of therapy resistance and tumor recurrence are still unresolved major challenges. This therefore underscores the need to identify novel tumor targets and develop corresponding therapies to supplement existing biologic and cytotoxic approaches so that a deeper and more sustained treatment responses could be achieved. The complement system is emerging as a potential novel target for cancer therapy. Data accumulated to date show that complement proteins, and in particular C1q and its receptors cC1qR/CR and gC1qR/p33/HABP1, are overexpressed in most cancer cells and together are involved not only in shaping the inflammatory tumor microenvironment, but also in the regulation of angiogenesis, metastasis, and cell proliferation. In addition to the soluble form of C1q that is found in plasma, the C1q molecule is also found anchored on the cell membrane of monocytes, macrophages, dendritic cells, and cancer cells, via a 22aa long leader peptide found only in the A-chain. This orientation leaves its 6 globular heads exposed outwardly and thus available for high affinity binding to a wide range of molecular ligands that enhance tumor cell survival, migration, and proliferation. Similarly, the gC1qR molecule is not only overexpressed in most cancer types but is also released into the microenvironment where it has been shown to be associated with cancer cell proliferation and metastasis by activation of the complement and kinin systems. Co-culture of either T cells or cancer cells with purified C1q or anti-gC1qR has been shown to induce an anti-proliferative response. It is therefore postulated that in the tumor microenvironment, the interaction between C1q expressing cancer cells and gC1qR bearing cytotoxic T cells results in T cell suppression in a manner akin to the PD-L1 and PD-1 interaction.

The role of complement in the tumor microenvironment

In order to enhance its growth and survival, a growing tumour can utilize a tumour-associated stroma, which can consist of recruited cells and host cells of the infiltrated tissue. Multiple stromal cell types as well as other cells and proteins make up the tumour-cell microenvironment. Proteins of the complement system—a key component of innate immunity—can interact with proteins of the tumour microenvironment to promote many hallmarks of cancer, including immunosuppression and angiogenesis. In this review article, Zhang et al. note that inappropriate activation of the complement system and deposition of complement proteins are common characteristics of various tumours. There is also evidence that overexpression of membrane-bound complement regulatory proteins (mCRPs) in cancer cells may protect them from complement-mediated attack and eventual cell lysis (see also {1}). This Recommendation is of an article referenced in a Faculty Review also written by Berhane Ghebrehiwet.

Infection with SARS-CoV-2 triggers the simultaneous activation of innate inflammatory pathways including the complement system and the kallikrein-kinin system (KKS) generating in the process potent vasoactive peptides that contribute to severe acute respiratory syndrome (SARS) and multi-organ failure. The genome of SARS-CoV-2 encodes four major structural proteins – the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. However, the role of these proteins in either binding to or activation of the complement system and/or the KKS is still incompletely understood. In these studies, we used: solid phase ELISA, hemolytic assay and surface plasmon resonance (SPR) techniques to examine if recombinant proteins corresponding to S1, N, M and E: (a) bind to C1q, gC1qR, FXII and high molecular weight kininogen (HK), and (b) activate complement and/or the KKS. Our data show that the viral proteins: (a) bind C1q and activate the classical pathway of complement, (b) bind FXII and HK, and activate the KKS in normal human plasma to generate bradykinin and (c) bind to gC1qR, the receptor for the globular heads of C1q (gC1q) which in turn could serve as a platform for the activation of both the complement system and KKS. Collectively, our data indicate that the SARS-CoV-2 viral particle can independently activate major innate inflammatory pathways for maximal damage and efficiency. Therefore, if efficient therapeutic modalities for the treatment of COVID-19 are to be designed, a strategy that includes blockade of the four major structural proteins may provide the best option.

Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SM^lowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SM^lowglu compared to MIC in SM with normal (2%) glucose (SM^nlglu). In SM^lowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SM^lowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SM^lowglu, it significantly decreased in the presence of FLC (12.5-fold) in SM^nlglu, but remained stable in SM^lowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SM^lowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1).

In addition to the ongoing COVID-19 pandemic, which has brought an unexpected global gloom, the beginning of this year was also particularly cruel to the complement field, as two of our greatest and dearest members—Sir Peter Lachmann and Prof

Nucleotide-binding oligomerization domain containing protein 2 (NOD2) is a cytosolic receptor. Both NOD2 and vasoactive intestinal peptide (VIP) are critical in regulation of immune and inflammatory response. Yao syndrome (YAOS, OMIM 617321) is an autoinflammatory disease associated with specified NOD2 mutations. Herein, we report a well-studied case of YAOS masquerading as mast cell disorder and neuroendocrine tumors to support the involvement of VIP in YAOS. For the first time, this case study suggests a potential relationship between NOD2 and VIP. This could provide a novel avenue for mechanistic study of NOD2-associated disease.